The turtle urinary bladder has proven to be a useful experimental model of the mammalian renal collecting duct. The bladder, like the collection duct, contains two major epithelial cell types, granular(G) cells, thought to possess the tissue's transepithelial Na transport function, and mitochondrial-rich(MR) cells, thought to contain the proton transport function. Recently, we have identified ATP-dependent H+ transport in cell-free vesicle preparations of the "native epithelium, that is, containing (presumably) membranes from both cell types. This was done using the fluorescent, pH-sensitive dye, acridine orange. It was decided to separate MR from G cells, prepare vesicles from each, and determine by the fluorescent dye technique whether active H+ transport does indeed originate in only one cell type. In this project, it is proposed: (1) To obtain enriched preparations of MR cells and separately, of G cells. Two independent methods will be used to separate MR from G cells: (a) centrifugation through stepwise density gradients of Ficoll and (b) free-flow electrophoresis. The former has been reported in the literature and confirmed by the applicant. The latter is a proven technique, in a variety of tissues, for separating different types of intact cells. Adequate separation will be defined quantitatively by marker enzyme activities and by known effects of inhibitors on MR cells and G cells. Separation will also be verified by light- and electron-microscopy. (2) To isolate enriched plasma membrane vesicles from each type of cell and determine which display active proton transport. Plasma membranes will be defined quantitatively as those enriched in plasma membrane enzyme markers and depleted in markers for critical subcellular organelles known to actively transport protons. Purification will be accomplished by conventional differential centrifugation in combination with sucrose density gradient centrifugation, free-flow electrophoresis, or both (as needed). (3) To functionally characterize active proton transport by the unequivocally identified urine-acidifying proton pump. Characterization will be in terms of the effects of known or possible inhibitors, determination of electrogenicity or electroneutrality, calibration of the maximal vesicular pH and PD formed, requirements for inorganic ions, and quantitation of the initial velocity of quenching of pH-sensitive dye fluorescence after addition of energetic substrates.